In the past few years, discoveries that others and we have made on the biophysics of neurotransmitter transporters have uncovered new modes of substrate and ion permeation. We discovered that cocaine and antidepressants block Na-dependent, NE-induced hNET currents. In addition to uncoupled current, there are channel modes of NE conduction, and voltage-dependent uptake is correlated with channel activity. PKC and Ca regulate both uptake and current. These results have encouraged us to emphasize three experimental areas of transporter fiinction that focus on coupling. How are uptake and current related? Where does coupling occur? What is the functional significance of coupled and uncoupled current? These questions form three Specific Aims: I Measure uptake/current coupling in hNET by patch clamp and micro-amperometry, II Investigate structural correlates for uptake/current coupling by mutagenesis, and III Construct a model "synapse" that tests acute regulation of uptake/current coupling. This competing renewal remains focused on hNET, which clears NE following its release in peripheral sympathetic and central noradrenergic synapses. Because neuronal activity influences NE uptake, we are interested in the acute regulation of hNET. To study these problems we use hNET-expressing HEK cells suitable for patch clamp, radioligand uptake, and biochemistry. We also use Xenopus oocytes to express wild type hNET and mutant clones for structure- function analysis, and we have introduced the frog equivalent of hNET, a recently cloned epinephrine transporter, fET. Finally, we have designed new protocols to study uptake and current in native preparations. Health significance: hNETs are used to load anti-hypertension drugs and antitumor agents. In ischemia hNET malfunction induces arrhythmia, and hNET-specific antidepressants cause cardiovascular complications. NE restores blood pressure and is used to stimulate the heart during cardiac arrest. Decreased uptake occurs in diabetes and excess NE increases platelet aggregation. Normal function and diseased states thus involve crucial regulatory mechanisms mediated by hNET. Our studies provide basic information about hNET function and loss of function relevant to human disease.